Methods of determining patient populations amenable to immunomodulatory treatment of cancer

ABSTRACT

The disclosure provides methods of determining patient populations amenable or suitable for immunomodulatory treatment of disease such as cancer by measuring the relative or absolute levels of T-cell sub-populations correlated with disease such as cancer.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the priority benefit under 35 U.S.C. § 119(e) ofProvisional U.S. Patent Application No. 62/146,043, filed Apr. 10, 2015,which is incorporated herein by reference in its entirety.

The work disclosed herein was supported, in part, by grant numberR01-CA136753 awarded by the National Cancer Institute. The federalgovernment has certain rights in the invention.

BACKGROUND

T-cells or T lymphocytes play a central role in cell-mediated immunity.They can be distinguished from other lymphocytes by the presence of aT-cell receptor (TCR) on the cell surface. T-cells mature in the thymus(although some also mature in the tonsils). There are several types of Tcells, and each type has a distinct function. The various types ofT-cells include T Helper cells (T_(H) cells), cytotoxic T-cells (CTLs),multiple sub-types of Memory T-cells, effector T cells (T_(eff)),regulatory T-cells (T_(reg)), and Natural Killer T-cells (NKT cells).

T helper cells (¾ cells) assist in the maturation of B cells into plasmacells and memory B cells, as well as in the activation of cytotoxic Tcells and macrophages. These ¾ cells, also known as CD4⁺ T-cells,express the CD4 glycoprotein on their surfaces. Helper T-cells becomeactivated when they are presented with peptide antigens by MHC class IImolecules, which are expressed on the surface of antigen-presentingcells or APCs. Once activated, Helper T-cells divide and secretecytokines that regulate the active immune response.

CD4 T effector cells (CD4 T_(eff)) are CD4⁺ FoxP3⁻ T cells that are notregulatory T cells and potentially have effector functions.

Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is an immune checkpointreceptor expressed on T cells that mediate inhibitory immune responsesin the early activation of naïve and memory T cells to maintain abalanced immune homeostasis (1) (2). CTLA-4 is found on the surface of Tcells, which are involved in the cellular immune response to foreignantigen. T-cells can be activated by stimulating the CD28 receptor onthe T-cell. T-cells can be inhibited by stimulating the CTLA-4 receptor,which acts as an “off” switch. CTLA-4 is a member of the immunoglobulinsuperfamily, which is expressed on the surface of Helper T cells andtransmits an inhibitory signal to T cells. CTLA-4 is similar to theT-cell co-stimulatory protein, CD28, and both molecules bind to CD80 andCD86, also called B7-1 and B7-2 respectively, on antigen-presentingcells. In contrast to the stimulatory signal transmitted by CD28, CTLA-4transmits an inhibitory signal to T-cells. T-cell activation through theT-cell receptor and CD28 leads to increased expression of CTLA-4, aninhibitory receptor for B7 molecules.

Programmed death 1 (PD-1) is a type I membrane protein of 268 aminoacids. PD-1 appears to negatively regulate immune responses, based onPD-1 knockout mice developing lupus-like glomerulonephritis and dilatedcardiomyopathy in C57BL/6 and BALB/c genetic backgrounds, respectively.T-cells stimulated by exposure to an anti-CD3 antibody that are exposedto a ligand of PD-1 (i.e., PD-L1) exhibit reduced T cell proliferationand IFN-γ secretion. These data indicate that PD-1 negatively regulatesT-cell responses.

Programmed death-ligand 1 (PD-L1, CD274, B7-H1) is a type I membraneprotein of 176 amino acids. PD-L1 is expressed on antigen-presentingcells (APCs), activated T cells, and a variety of tissues. PD-L1knockout mice demonstrated increased CD4 and CD8 T cell responsesincluding increased levels of cytokines. This data suggests that PD-L1negatively regulates T cells and play a role in T cell tolerance.

The foregoing aspects of the immune system and the many other aspects ofthe immune system have been extensively studied for years as biologistsand medical professionals have recognized the role played by the immunesystem in resisting disease and in combatting infection. Efforts toharness the considerable potential of the immune system to treat orprevent disease have led to the development of a number of biologiesshowing promise in the clinic. Those efforts, however, have requiredsignificant capital input and time expenditures due to care that must betaken in developing a treatment for humans, the number of failures, andthe uneven response profiles of patients receiving treatment.

For all of the foregoing reasons, a need continues to exist in the artfor materials and methods that yield effective immunomodulatorytherapeutics, such as biologies, in an efficient manner and that moreprecisely target amenable or suitable patient populations for treatmentor prevention.

SUMMARY

The disclosure provides methods for determining whether cancer patientsare amenable or suitable for immunomodulatory treatment of the cancer byquantifying or assessing PD-1⁺ and/or CTLA-4⁺ and/or PD-L1⁺ T-cellsub-types by measuring the expression of one or two or three markers,comparing the relative expression levels and determining whether thepatients are amenable to such treatment based on those expressionlevels. The methods allow tailoring of potentially efficacious cancertreatment to precisely those patients best-situated to benefit from thecostly treatments. The increased efficiency in cancer treatments willmake positive contributions to cancer treatment while reducing theoverall costs associated with treating the various forms of thisdisease.

In one aspect, the disclosure provides a method of determining that acancer patient population is amenable to immunomodulatory inhibition ofthe growth of a cancer cell comprising (a) obtaining a sample from acancer patient; (b) quantifying the percentage of PD-1⁺ in CD4⁺ T_(e)_(ff) or total CD4⁺ T cells in the sample; (c) assessing the percentageof PD-1⁺ in CD4⁺ T_(eff) or total CD4⁺ T cells from a non-canceroussubject; (d) comparing the percentage of PD-1⁺ in CD4⁺ T_(eff) or totalCD4⁺ T cells in (b) to the percentage of PD-1⁺ in CD4⁺ T_(eff) or totalCD4⁺ T cells in (c); and (e) determining that the cancer patient isamenable to immunomodulatory inhibition of the cancer cell when thepercentage of PD-1⁺ in CD4⁺ T_(eff) or total CD4⁺ T cells in (b) is nogreater than the percentage of PD-1⁺ in CD4⁺ T_(eff) or total CD4⁺ Tcells in (c). The method may further comprise contacting the sample withan anti-PD-1 antibody product and isolating the PD-1⁺ CD4⁺ T_(eff) ortotal CD4⁺ T cells in the sample.

In one aspect, the disclosure provides a method of determining that acancer patient population is amenable to immunomodulatory inhibition ofthe growth of a cancer cell comprising: (a) obtaining a sample from acancer patient; (b) quantifying the percentage of CTLA-4⁺ in CD4⁺ T_(e)_(ff) or total CD4⁺ T cells in the sample; (c) assessing the percentageof CTLA-4⁺ in CD4⁺ T_(e) _(ff) or total CD4⁺ T cells from anon-cancerous subject; (d) comparing the percentage of CTLA-4⁺ in CD4⁺T_(eff) or total CD4⁺ T cells in (b) to the percentage of CTLA-4⁺ inCD4⁺ T_(e) _(ff) or total CD4⁺ T cells in (c); and (e) determining thatthe cancer patient is amenable to immunomodulatory inhibition of thecancer cell when the percentage of CTLA-4⁺ in CD4⁺ T_(e) _(ff) or totalCD4⁺ T cells in (b) is greater than the percentage of CTLA-4⁺ in CD4⁺T_(eff) or total CD4⁺ T cells in (c). The method may further comprisecontacting the sample with an anti-CTLA-4 antibody product and isolatingthe CTLA-4⁺ CD4⁺ T_(e) _(ff) or total CD4⁺ T cells in the sample.

In one aspect, the disclosure provides a method of determining that acancer patient population is amenable to immunomodulatory inhibition ofthe growth of a cancer cell comprising: (a) obtaining a sample from acancer patient; (b) quantifying the percentage of PD-L1⁺ in CD4⁺ T_(e)_(ff) or total CD4⁺ T cells in the sample; (c) assessing the percentageof PD-L1⁺ in CD4⁺ T_(e) _(ff) or total CD4⁺ T cells from a non-canceroussubject; (d) comparing the percentage of PD-L1⁺ in CD4⁺ T_(eff) or totalCD4⁺ T cells in (b) to the percentage of PD-L1⁺ in CD4⁺ T_(eff) or totalCD4⁺ T cells in (c); and (e) determining that the cancer patient isamenable to immunomodulatory inhibition of the cancer cell when thepercentage of PD-L1⁺ in CD4⁺ T_(e) _(ff) or total CD4⁺ T cells in (b) isno greater than the percentage of PD-L1⁺ in CD4⁺ T_(ef) _(f) or totalCD4⁺ T cells in (c). The method may further comprise contacting thesample with an anti-PD-L1 antibody product and isolating the PD-L1⁺ CD4⁺T_(e) _(ff) or total CD4⁺ T cells in the sample.

Another aspect according to the disclosure is a method of identifying acancer patient amenable to immunomodulatory inhibition of cancer cellgrowth comprising: (a) obtaining a sample from a cancer patient; (b)contacting the cancer patient sample with at least one antibody productthat specifically binds a marker selected from the group consisting ofPD-1, PD-L1 and CTLA-4; (c) assessing the relative level of T-cellsexhibiting the marker in the CD4⁺ T_(e) _(ff) or total CD4⁺ T cellpopulation in the cancer patient sample by measuring the level of CD4⁺T_(e) _(ff) or total CD4⁺ T cells expressing the marker in the CD4⁺T_(e) _(ff) or total CD4⁺ T cell population in the cancer patientsample; (d) quantifying the relative level of T_(e) _(ff) or total CD4⁺T cells exhibiting the marker in the CD4⁺ T_(e) _(ff) or total CD4⁺ Tcell population of a sample from a non-cancerous subject by measuringthe level of T_(e) _(ff) or total CD4⁺ T cells expressing the marker inthe CD4⁺ T_(e) _(ff) or total CD4⁺ T cell population in the sample fromthe non-cancerous subject; (e) comparing the relative level of CD4⁺T_(e) _(ff) or total CD4⁺ T cells exhibiting the marker in the CD4⁺T_(e) _(ff) or total CD4⁺ T cell population of the cancer patient sampleto the relative level of CD4⁺ T_(e) _(ff) or total CD4⁺ T cellsexhibiting the marker in the CD4⁺ T_(e) _(ff) or total CD4⁺ T cellpopulation of a sample from the non-cancerous subject; and (f)determining the cancer patient to be amenable to anti-CTLA-4immunomodulatory inhibition of the cancer cell when the relative levelof CD4⁺ T_(e) _(ff) or total CD4⁺ T cells exhibiting the marker in theCD4⁺ T_(e) _(ff) or total CD4⁺ T cell population in the cancer patientsample is similar or reduced relative to the level of CD4⁺ T_(e) _(ff)or total CD4⁺ T cells exhibiting the marker in the CD4⁺ T_(e) _(ff) ortotal CD4⁺ Tc ell population in the sample of the non-cancerous subjectif the marker is PD-1 or PD-L1, or determining the cancer patient to beamenable to anti-CTLA-4 immunomodulatory inhibition of the cancer cellwhen the relative level of CD4⁺ T_(eff) or total CD4⁺ T cells exhibitingthe marker in the CD4⁺ T_(eff) or total CD4⁺ T cell population in thecancer patient sample is increased relative to the level of CD4⁺ T_(e)_(ff) or total CD4⁺ T cells exhibiting the marker in the CD4⁺ T_(e)_(ff) or total CD4⁺ T cell population in the sample of the non-canceroussubject if the marker is CTLA-4. When referring to relative levels ofCD4⁺ T_(e) _(ff) or total CD4⁺ T cells being similar or reduced relativeto the level of CD4⁺ T_(e) _(ff) or total CD4⁺ T cells in anothersample, the level in the first sample is about the same or less than thelevel in the second sample, i.e., the level of CD4⁺ T_(e) _(ff) or totalCD4⁺ T cells in the first sample is no greater than the level of CD4⁺T_(e) _(ff) or total CD4⁺ T cells in the second sample.

Another aspect is drawn to a method comprising: (a) measuring PD-1,PD-L1 and CTLA-4 expression in CD4⁺ T_(e) _(ff) or total CD4⁺ T cells ina biological sample; and (b) detecting a similar or reduced level ofPD-1 expression or PD-L1 expression, or an increased level of CTLA-4expression, or any combination thereof, in the sample relative to thelevel in a non-cancerous subject. A related aspect of the disclosureprovides a method comprising: (a) measuring PD-1, PD-L1, or CTLA-4expression by CD4⁺ T_(eff) or total CD4⁺ T cells in a biological samplethat comprises T cells from a mammalian subject having cancer; and (b)detecting a similar or reduced level of PD-1-expressing CD4⁺ T_(e) _(ff)or total CD4⁺ T cells, a similar or reduced level of PD-L1-expressingCD4⁺ T_(e) _(ff) or total CD4⁺ T cells, or an elevated level ofCTLA-4-expressing T cells in the sample relative to the level in anon-cancerous subject. Yet another related aspect of the disclosure isdirected to a method comprising: (a) measuring PD-1, PD-L1 or CTLA-4expression by CD4⁺ T_(e) _(ff) or total CD4⁺ T cells in a biologicalsample that comprises T cells from a mammalian subject having cancer;and (b) identifying the subject as amenable to anti-CTLA-4immunomodulatory therapy based on the measurement of the PD-1, PD-L1 orCTLA-4 expression by CD4⁺ T_(e) _(ff) or total CD4⁺ T cells in thebiological sample. In some embodiments of these aspects of thedisclosure, PD-1 expression, or PD-L1 expression, or CTLA-4 expressionis measured.

Embodiments of any of the foregoing aspects of the disclosure providemethods wherein the PD-1⁺ CD4⁺ T_(e) _(ff) or total CD4⁺ T cells arepresent in the sample at a level no greater than 106 cells permicroliter of sample or at a percentage no higher than 21% of the CD4⁺T_(e) _(ff) or total CD4⁺ T cells in the sample of a patient amenable toimmunomodulatory inhibition of cancer cell growth. In some embodiments,the marker is CTLA-4, such as embodiments wherein the CTLA-4⁺ CD4⁺ T_(e)_(ff) or total CD4⁺ T cells are present in the sample at a level no lessthan 99 cells per microliter of sample or at a percentage no less than15.6% of the CD4⁺ T_(e) _(ff) or total CD4⁺ T cells in the sample of apatient amenable to immunomodulatory inhibition of cancer cell growth.In some embodiments, the marker is PD-L1, such as embodiments whereinthe PD-L1⁺ CD4⁺ T_(e) _(ff) or total CD4⁺ T cells are present in thesample at a level no greater than 200 cells per microliter of sample orat a percentage no higher than 23.5% of the CD4⁺ T_(e) _(ff) or totalCD4⁺ T cells in the sample of a patient amenable to immunomodulatoryinhibition of cancer cell growth.

Embodiments of any of the foregoing aspects of the disclosure providemethods wherein the sample is a tumor sample or a blood sample. By tumorsample is meant any abnormal benign or malignant growth of tissue thatarises from uncontrolled cellular proliferation. A “tumor” includes oneor more tumor cells and/or one or more tumor-infiltrating lymphocytes.The “blood” in a blood sample is understood to include whole blood,serum, and isolated blood cells, including peripheral blood mononuclearcells, other leukocytes, erythrocytes and platelets. Relative to tumorsamples, blood samples are much easier to obtain, requiring simpler,less invasive and less risky procedures to the patient that can also beperformed quicker and at reduced cost. These advantages of manipulatingblood samples are apparent when the markers are potential biomarkersthat have prognostic or predictive value.

Analogously, embodiments of any of the foregoing aspects of thedisclosure provide methods wherein the antibody product is a polyclonalantibody, a monoclonal antibody, an antigen-binding antibody fragmentthereof, a hybrid antibody, a chimeric antibody, a CDR-grafted antibody,a single-chain antibody, a single-chain variable fragment, a Fabantibody fragment, a Fab′ antibody fragment, a F(ab′)2 antibodyfragment, a linear antibody, a bi-body, a tri-body, a diabody, apeptibody, a bispecific antibody, a bispecific T-cell engaging (BiTE)antibody, or a chimeric antibody receptor. Exemplary antibody productsare an anti-PD-1 antibody product or an anti-PD-L1 antibody product oran anti-CTLA-4 antibody product. Exemplary cancer cells are anadenocarcinoma cell, a castration-resistant prostate cancer cell, amelanoma cell, a Head-and-Neck cancer cell, a lung cancer cell, a kidneycancer cell, a bladder cancer cell, a gastric cancer cell, a colorectalcancer cell, an ovarian cancer cell, a hepatocellular cancer cell, ahepatobiliary cancer cell, or a breast cancer cell, although thedisclosure contemplates that any cancer cell known in the art issuitable for use in the methods. In some embodiments, the cancer cell isa an adenocarcinoma cell, a castration-resistant prostate cancer cell, amelanoma cell.

In an aspect related to each of the foregoing aspects, the disclosureprovides a method as described above, further comprising (a) quantifyingthe granzyme B level in the CD4⁺ T_(eff) or total CD4⁺ T cells or in thePD-1⁺ CD4⁺ T_(eff) or PD-1⁺ total CD4⁺ T cells in the cancer patientsample; and (b) determining that the cancer patient is amenable toimmunomodulatory inhibition of the cancer cell when the granzyme B levelis elevated relative to the granzyme B level in a sample from anon-cancerous subject. In some embodiments of this aspect, the cancerpatient is determined to be amenable to immunomodulatory treatment whenthe percentage of CTLA-4⁺ CD4⁺ T_(eff) or CTLA-4⁺ total CD4⁺ T cells inCD4⁺ T_(eff) or total CD4⁺ T cells is at least 15.6% or the number ofCTLA-4⁺ CD4⁺ T_(eff) or CTLA-4⁺ total CD4⁺ T cells is at least 99 cellsper microliter of sample.

Another aspect related to each of the foregoing aspects of thedisclosure is drawn to a method as described above, further comprisingadministering a therapeutically effective amount of an anti-CTLA-4immunomodulatory agent to the subject identified as exhibiting the lowerlevel of PD-1-expressing CD4⁺ T_(eff) or total CD4⁺ T cells, or thelower level of PD-L1-expressing CD4⁺ T_(eff) or total CD4⁺ T cells, orthe elevated level of CTLA-4-expressing CD4⁺ T_(eff) or total CD4⁺ Tcells.

Yet another aspect of the disclosure is drawn to a use of PD-1expression, PD-L1 expression, or CTLA-4 expression in CD4⁺ T_(eff) ortotal CD4⁺ T cells in a mammalian subject having cancer for identifyinga subject amenable to anti-CTLA-4 immunomodulatory therapy.

Still another aspect of the disclosure is a method of treatment of amammalian subject having cancer comprising: (a) administering atherapeutically effective amount of an anti-CTLA-4 immunomodulatorytherapy to a subject identified as amenable to anti-CTLA-4immunomodulatory treatment based on having a lower level ofPD-1-expressing CD4⁺ T_(eff) or PD-1⁺ total CD4⁺ T cells, or a lowerlevel of PD-L1-expressing CD4⁺ T_(eff) or PD-1⁺ total CD4⁺ T cells, oran elevated level of CTLA-4-expressing CD4⁺ T_(eff) or PD-1⁺ total CD4⁺T cells, or any combination thereof, as measured in a biological sampleof the subject.

Another aspect of the disclosure is a cancer treatment protocolcomprising: (a) measuring PD-1 or PD-L1 or CTLA-4 expression by CD4⁺T_(eff) or total CD4⁺ T cells in a biological sample that comprises CD4⁺T_(eff) or PD-1⁺ total CD4⁺ T cells from a mammalian subject; and (b)administering a therapeutically effective amount of an anti-CTLA-4immunomodulatory therapy to a subject identified as amenable toimmunomodulatory treatment of cancer on the basis of a lower level ofPD-1-expressing CD4⁺ T_(eff) or total CD4⁺ T cells, a lower level ofPD-L1-expressing CD4⁺ T_(eff) or total CD4⁺ T cells, or an elevatedlevel of CTLA-4-expressing CD4⁺ T_(eff) or total CD4⁺ T cells in thesample.

The disclosure provides another aspect drawn to a method of inhibitingthe growth of a cancer cell in a targeted population of cancer patientscomprising (a) obtaining a sample from a cancer patient; (b) contactingthe sample with an antibody product specifically binding to a T-cellmarker selected from the group consisting of CTLA-4, PD-L1 and PD-1; (c)measuring the marker bound by the antibody product; (d) identifying thecancer patient as amenable to anti-CTLA-4 immunomodulatory inhibition ofcancer cell growth when (i) there are more CTLA-4⁺ CD4⁺ T_(eff) or totalCD4⁺ T cells in the CD4⁺ T_(eff) or total CD4⁺ T cell population of thepatient sample than in the CD4⁺ T_(e) _(ff) or total CD4⁺ T cellpopulation of a sample from a non-cancerous subject, or there are fewerPD-1⁺ CD4⁺ T_(eff) or total CD4⁺ T cells in the CD4⁺ T_(eff) or totalCD4⁺ T cell population of the patient sample than in the CD4⁺ T_(e)_(ff) or total CD4⁺ T cell population of a sample from a non-canceroussubject, or there are fewer PD-L1⁺ CD4⁺ T_(e) _(ff) or total CD4⁺ Tcells in the CD4⁺ T_(e) _(ff) or total CD4⁺ T cell population of thepatient sample than in the CD4⁺ T_(e) _(ff) or total CD4⁺ T cellpopulation of a sample from a non-cancerous subject; or (ii) thepercentage of CD4⁺ T_(e) _(ff) or total CD4⁺ T cells comprising themarker in the sample is at least 15.6% or the absolute quantity of CD4⁺T_(e) _(ff) or total CD4⁺ T cells comprising the marker in the sample isat least 99 per μï of sample, if the marker is CTLA-4⁺, or (iii) thepercentage of CD4⁺ T_(e) _(ff) or total CD4⁺ T cells comprising themarker in the sample is no greater than 21% or the absolute quantity ofCD4⁺ T_(e) _(ff) or total CD4⁺ T cells comprising the marker in thesample is no greater than 106 per μï of sample, if the marker is PD-1⁺,or (iv) the percentage of CD4⁺ T_(e) _(ff) or total CD4⁺ T cellscomprising the marker in the sample is no greater than 23.5% or theabsolute quantity of CD4⁺ T_(e) _(ff) or total CD4⁺ T cells comprisingthe marker in the sample is no greater than 200 per μï of sample, if themarker is PD-L1⁺; and (e) administering a therapeutically effectiveamount of an immunomodulatory agent to the patient amenable toimmunomodulatory inhibition of cancer cell growth. In some embodimentsof this aspect, the sample is a tumor sample or a blood sample.

In some embodiments of this aspect of the disclosure, the antibodyproduct is a polyclonal antibody, a monoclonal antibody, anantigen-binding antibody fragment thereof, a hybrid antibody, a chimericantibody, a CDR-grafted antibody, a single-chain antibody, asingle-chain variable fragment, a Fab antibody fragment, a Fab′ antibodyfragment, a F(ab′)2 antibody fragment, a linear antibody, a bi-body, atri-body, a diabody, a peptibody, a bispecific antibody, a bispecificT-cell engaging (BiTE) antibody, or a chimeric antibody receptor, suchas an antibody product that is a CTLA-4-binding antibody fragment. Anexemplary antibody product for use in this aspect of the disclosure isipilimumab. Exemplary cancer cells growth-inhibited by methods of thedisclosure are cells of an adenocarcinoma, a castration-resistantprostate cancer, a melanoma, a Head-and-Neck cancer, a lung cancer, akidney cancer, a bladder cancer, a gastric cancer, a colorectal cancer,an ovarian cancer, a hepatocellular cancer, a hepatobiliary cancer, andbreast cancer, although the disclosure contemplates that any cancer cellknown in the art is suitable for use in the methods. In someembodiments, the cancer cell is an adenocarcinoma cell, acastration-resistant prostate cancer cell, or a melanoma cell.

Other features and advantages of the disclosure will be betterunderstood by reference to the following detailed description, includingthe drawing and the examples.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Ipilimumab (anti-CTLA-4) treatment schedule. A, Metastaticcastration resistant prostate cancer (mCPRC) patients were treated every4 weeks with anti-CTLA-4 administered on day 1 of every cycle and GM-CSFadministered at 250 μg/m²/dose from day 1 to day 14 daily of everycycle. Escalating doses were given to each cohort with an expansioncohort at 3 mg/kg/dose. B, Metastatic melanoma patients were treatedevery 3 weeks with anti-CTLA-4 administered at 10mg/kg/dose on day 1 ofevery cycle and GM-CSF administered at 125 μg/m²/dose from day 1 to day14 daily of every cycle.

FIG. 2. Clinical outcomes of 42 metastatic castration resistant prostatecancer (mCRPC) patients in a Phase 1b ipilimumab (anti-CTLA-4) andGM-CSF clinical trial. A, Waterfall plot of the maximum percentagechange in PSA from baseline of each patient until nadir or off study.Dashed line shows 50% decline in PSA. B, Spider plot shows change in PSAwith time from baseline of each patient until nadir or off study. Dashedline shows 50% decline in PSA. C, Graph showing the duration of studytreatment, duration of response, time to disease progression, and timeto at least 50% decline in PSA for each patient. D, Overall survivalcurve for all patients as of analysis on the censor date. Dotted linesbelow and above the survival curve (solid line) show lower and upper 95%confidence intervals (CI) respectively. Vertical tick marks indicate OSof patients who were still alive as of the censor date.

FIG. 3. Clinical outcomes of 21 metastatic melanoma patients in a PhaseII ipilimumab (anti-CTLA-4) and GM-CSF clinical trial. A, Overallsurvival curve for all patients as of analysis on the censor date.Dotted lines below and above the survival curve (solid line) show lowerand upper 95% confidence intervals (CI) respectively. Vertical tickmarks indicate OS of patients who were still alive as of the censordate. B, Kaplan-Meier plots of treated melanoma patients separated bypresence of complete response (CR) plus partial response (PR) plusstable disease (SD) compared to patients with progressive disease (PD).Sum of patients with CR, PR or SD are hence denoted as responders (R)and sum of patients with PD are hence denoted as non-responders (NR).

FIG. 4. Surface PD-1 expression of CD4 T_(eff) cells and CD8 T cells formCRPC patients. A, Flow cytometry was used to assess PD-1 expression byCD4 T_(eff) cells and CD8 T cells. Percentage of PD-1 positive cells inantibody-stained sample was gated based on isotype-containing controls.Shaded histograms denote isotype controls; open histograms denotestained samples. B and C, Time course of percentage of CD4 T_(eff) andCD8 T cells that express PD-1 respectively. Connected dots show timecourse of the same patient. D and E, Time course of absolute counts ofCD4 T_(eff) and CD8 T cells that express PD-1 respectively. F and G, Boxplots of percentage of CD4 T_(eff) and CD8 T cells that express PD-1respectively for long-term (L) and short-term (S) survivors at each timepoint. H and I, Box plots of absolute counts of CD4 T_(eff) and CD8 Tcells that express PD-1 respectively for long-term (L) and short-term(S) survivors at each time point. Whiskers show minimum and maximumlevels. *p-value<0.05, **p-value<0.01, ***p-value<0.001.

FIG. 5. Total CTLA-4⁺ expression in total CD4 and CD8 T cells for mCRPCpatients. A, Flow cytometry was used to assess CTLA-4 expression by CD4T cells and CD8 T cells. Percentage of CTLA-4 positive cells inantibody-stained sample was gated based on isotype-containing controls.Shaded histograms denote isotype controls; open histograms denotestained samples. B and C, Time course of percentage of CD4 T and CD8 Tcells that express CTLA-4 respectively. Connected dots show time courseof the same patient. D and E, Time course of absolute counts of CD4 Tand CD8 T cells that express CTLA-4 respectively. F and G, Box plots ofpercentage of CD4 T and CD8 T cells that express CTLA-4 respectively forlong-term (L) and short-term (S) survivors at each time point. H and I,Box plots of absolute counts of CD4 T and CD8 T cells that expressCTLA-4 respectively for long-term (L) and short-term (S) survivors ateach time point. Whiskers show minimum and maximum levels. *p-value<0.05, **p-value<0.01, ***p-value<0.001.

FIG. 6. Surface PD-L1 expression of CD4 cells and CD8 T cells formetastatic melanoma patients. A, Flow cytometry was used to assess PD-L1expression by CD4 T cells and CD8 T cells. Percentage of PD-L1 positivecells in antibody-stained sample was gated based on isotype-containingcontrols. Shaded histograms denote isotype controls; open histogramsdenote stained samples. B and C, Time course of percentage of CD8 T andCD4 T cells that express PD-L1 respectively. Connected dots show timecourse of the same patient. D and E, Time course of absolute counts ofCD8 T and CD4 T cells that express PD-L1 respectively. F and G, Boxplots of percentage of CD4 T and CD8 T cells that express PD-L1respectively for responders (R) and non-responders (NR) at each timepoint. H and I, Box plots of absolute counts of CD8 T and CD4 T cellsthat express PD-L1 respectively for responders (R) and non-responders(NR) at each time point. Whiskers show minimum and maximum levels.*p-value<0.05, **p-value<0.01, ***p-value<0.001, ****p-value<0.0001.

FIG. 7. Levels of circulating lymphocytes, CD4 T_(eff) cells and CD8 Tcells with treatment for mCRPC patients. A, Time course of absolutelymphocyte counts for assessed patients at week 0 (pre-treatment), week4 (cycle 1), and week 8 (cycle 2) of treatment. Connected dots show timecourse of the same patient. B, Box plots of absolute lymphocyte countsfor long-term (L) and short-term (S) survivors at each time point. C andG, Time course of percentage of CD4 T_(e) _(ff) cells and CD8 T cells oftotal lymphocytes, respectively. Connected dots show time course of thesame patient. E and I, Time course of absolute counts of CD4 T_(e) _(ff)cells and CD8 T cells respectively. D and H, Box plots of percentage ofCD4 T_(eff) cells and CD8 T cells of total lymphocytes respectively forlong-term (L) and short-term (S) survivors at each time point. F and J,Box plots of absolute counts of CD4 T_(e) _(ff) cells and CD8 T cellsrespectively for long-term (L) and short-term (S) survivors at each timepoint. Whiskers show minimum and maximum levels. *p-value<0.05,**p-value<0.01, ***p-value<0.001.

FIG. 8. Levels of circulating lymphocytes, CD4 T cells and CD8 T cellswith treatment for metastatic melanoma patients. A, Time course ofabsolute lymphocyte counts for assessed patients at week 0(pre-treatment), week 3 (cycle 1), and week 6 (cycle 2) of treatment.Connected dots show time course of the same patient. B, Box plots ofabsolute lymphocyte counts for responders plus SD (R) and non-responders(N) at each time point. C and G, Time course of percentage of CD4 Tcells and CD8 T cells of total lymphocytes respectively. Connected dotsshow time course of the same patient. E and I, Time course of absolutecounts of CD4 T cells and CD8 T cells respectively. D and H, Box plotsof percentage of CD4 T cells and CD8 T cells of total lymphocytesrespectively for responders plus SD (R) and non-responders (N) at eachtime point. F and J, Box plots of absolute counts of CD4 T cells and CD8T cells respectively for responders plus SD (R) and non-responders (N)at each time point. Whiskers show minimum and maximum levels. *p-value<0.05, **p-value<0.01, ***p-value<0.001.

FIG. 9. Comparison of PD-1, CTLA-4 and PD-L1 expression levels betweencancer-free controls and cancer patients at baseline. A, Box plots ofpercentage of CD4 T_(e) _(ff) cells that express PD-1 for cancer-freecontrols (Controls), long-term (L) and short-term (S) survivors formCRPC. B, Box plots of percentage of total CD4 T cells that expressCTLA-4 for cancer-free controls (Controls), long-term (L) and short-term(S) survivors for mCRPC. C, Box plots of percentage of CD4 T_(e) _(ff)cells that express PD-1 for cancer-free controls (Controls), responders(R) and non-responders (N) for metastatic melanoma. D, Box plots ofpercentage of total CD4 T cells that express PD-L1 for cancer-freecontrols (Controls), responders (R) and non-responders (N) formetastatic melanoma. Whiskers show minimum and maximum levels.*p-value<0.05, **p-value<0.01, ***p-value<0.001.

FIG. 10. Comparison of overall survival of patients separated by levelsof immune subsets a designated in the legend. A and B, Kaplan-Meierplots of percentage of CD4 T_(eff) cells that expressed PD-1 andabsolute counts of PD-1⁺ CD4 T_(eff) cells respectively, separated bycutoff levels designated in the legend. C and D, Kaplan-Meier plots ofpercentage of CD4 T cells that expressed CTLA-4 and absolute counts ofCTLA-4⁺ CD4 T cells respectively, separated by cutoff levels designatedin the legend. E and F, Kaplan-Meier plots of percentage of CD4 T cellsthat expressed PD-L1 and absolute counts of PD-L1⁺ CD4 T cellsrespectively, separated by cutoff levels designated in the legend.

FIG. 11. A, Cytokine expression and surface markers of PD-1⁺ and PD-ΓCD4 T cells. Representative flow cytometry diagrams showing percentagesof IFNy, IL-4, granzyme B, CD49b, and Lag-3 expression in PD-1⁺ and PD-ΓCD4 T cells for one out of three healthy donors and one out of six mCRPCpatients. Pre-treatment PBMC from cancer patients were used. B, Cytokineexpressions and surface markers of PD-1⁺ and PD-Γ CD8 T cells.Representative flow cytometry diagrams showing percentages of IFNy,IL-4, granzyme B, CD49b, and Lag-3 expression in PD-1⁺ and PD-Γ CD8 Tcells for one out of three healthy donors and one out of six mCRPCpatients. Pre-treatment PBMC from cancer patients were used. For IFNyand IL-4 staining, PBMC were stimulated with PMA and ionomycin for 4hours in culture at 37° C. with Brefeldin A added at the last 2 hours ofincubation. For granzyme B, CD49b, and Lag-3 staining, unstimulated PBMCwere used.

FIG. 12. Baseline characteristics for mCPRC patients. A, B, C, and D,Box plots of age, baseline PSA levels, LDH levels, and months on studyrespectively for long-term (L) and short-term (S) survivors. Whiskersshow minimum and maximum levels. E, F, G, H, I, and J, Bar graphsshowing the number of patients with the attributes designated in thelegends for ECOG, Gleason score, prior RP, prior radiation, subsequenttherapies, and clinical responses, respectively, for long-term (L) andshort-term (S) survivors.

FIG. 13. Baseline characteristics for metastatic melanoma patients. A,Box plot of age for responders (R) and non-responders (NR). Whiskersshow minimum and maximum levels. B, C, D, E, and F, Bar graphs showingthe number of patients with the attributes designated in the legends forsex, tumor stage, prior therapy, immune adverse events, and priorsystemic therapy respectively for responders (R) and non-responders(NR).

FIG. 14. Surface PD-1 expression of CD4 T_(eff) cells and CD8 T cellsfor metastatic melanoma patients. A, Flow cytometry was used to assessPD-1 expression by CD4 T_(eff) cells and CD8 T cells. Percentage ofPD-1-positive cells in antibody-stained sample was gated based onisotype-containing controls. Shaded histograms denote isotype controls;open histograms denote stained samples. B and C, Time course ofpercentage of CD4 T_(eff) and CD8 T cells that express PD-1,respectively. Connected dots show time course of the same patient. D andE, Time course of absolute counts of CD4 T_(eff) and CD8 T cells thatexpress PD-1, respectively. F and G, Box plots of percentage of CD4T_(eff) and CD8 T cells that express PD-1, respectively, for responders(R) and non-responders (NR) at each time point. H and I, Box plots ofabsolute counts of CD4 T_(eff) and CD8 T cells that express PD-1,respectively, for responders (R) and non-responders (NR) at each timepoint. Whiskers show minimum and maximum levels. *p-value<0.05,**p-value<0.01, ***p-value<0.001.

DETAILED DESCRIPTION

Cytotoxic T-Lymphocyte-associated antigen 4 (CTLA-4) blockade can inducetumor regression and improve survival in cancer patients. This treatmentcan enhance adaptive immune responses without an exogenous vaccine, butthe immunologic parameters associated with improved clinical outcome inprostate cancer patients are not established. Ipilimumab is a fullyhumanized monoclonal antibody targeting CTLA-4 that is FDA approved forthe treatment of melanoma. In two phase III studies in advancedmelanoma, ipilimumab was shown to significantly prolong overall survival(OS) (3) (4). In the pivotal trial, melanoma patients were treated withipilimumab plus gplOO (a melanoma peptide vaccine), ipilimumab alone orgplOO alone (3). The median overall survivals were 10.0, 10.1, and 6.4months respectively. Although improvement in median overall survival wasmodest, a subset of patients was observed in these and other melanomaclinical trials to have durable long-term survival benefit (5) (6).Notably, long-term survival can happen without accompanying objectiveresponse. Additionally, treatment with ipilimumab plus sargramostim(GM-CSF) resulted in improved median OS and lower toxicity compared toipilimumab alone (17.5 months versus 12.7 months) in a phase II clinicaltrial with unresectable melanoma (7).

A phase III clinical trial for metastatic castration resistant prostatecancer (mCRPC) treated with ipilimumab versus placebo after radiotherapyreported no significant difference in OS between the two groups (8).Median OS was 11.2 months for the ipilimumab treated group and 10.0months for the placebo group. However, it was observed that the hazardratio (HR) decreased over time, showing that ipilimumab associated withbetter survival at later time points. HR for 0-5 months was 1.46 (95% CI1.10-1.95) and for beyond 12 months was 0.6 (95% CI 0.43-0.86).Treatment with ipilimumab also improved progression-free survivalcompared to placebo (median 4.0 versus 3.1 months; HR 0.70; p-value lessthan 0.001). Post hoc analysis showed that patients who have no visceralmetastases benefited more from ipilimumab treatment than those that dohave visceral metastases (HR 1.64; p-value=0.0056).

Disclosed herein is an up-dated long-term follow-up of a phase 1b doseescalation trial in patients with metastatic, castration resistantprostate cancer (mCRPC) was performed combining ipilimumab (anti-CTLA-4)and sargramostim (GM-CSF). Patients were followed clinically forresponse and overall survival, and for immunomodulation of circulating Tcells. Of the 42 mCRPC enrolled patients, five patients had PSA declinesof at least 50% with associated radiographic responses in two patientsfrom the groups treated with at least 3 mg/kg of ipilimumab. Long-termfollow-up demonstrated that 16 patients (38%) had overall survival ofgreater than 30 months, of whom four patients had PSA declines of atleast 50% or objective responses. The combination of GM-CSF andipilimumab can induce prolonged survival in a subset of patients who didnot have PSA or objective responses. Clinical responses were bothimmediate and delayed. Two patients were still alive with overallsurvivals of 4.8 and 7 years as of censored date on Oct. 21 2014. One ofthese patients had a continued PSA response without additional therapy.

Disclosed herein are the clinical results of a phase II trial inpatients with metastatic melanoma was performed combining ipilimumab(anti-CTLA-4) and sargramostim (GM-CSF). Of the 22 metastatic melanomapatients, one patient received only 1 cycle of ipilimumab andsargramostim and was not included in subsequent analysis. Of the 21remaining patients, one patient had complete response (CR), 6 patientshad partial response (PR), 1 patient had stable disease (SD), and 13patients had progressive disease (PD). These clinical responses werefound to associate with overall survival. As of censored date on Feb. 122015, 10 patients were still alive.

Immune subsets in peripheral blood mononuclear cells (PBMC) wereevaluated for 23 out of 42 mCRPC patients whom were treated withipilimumab at 3 mg/kg/dose or greater and sargramostim at 250μg/m²/dose. Immune subsets were examined in PBMC from baseline, aftercycle 1 and after cycle 2 of treatment with flow cytometry. As thenumber of clinical responses observed were low, comparison of immunesubsets were made between patients who had overall survival greater thanthe median (23.6 months) for the group (long-term survivors, LTS, OSrange: 25.4 months-99.7 months) (n=11)) versus patients who had overallsurvival less than the median for the group (short-term survivors, STS,OS range: 1.9 months-22.4 months) (n=12)).

Immune subsets in PBMC were evaluated for all 2 1 metastatic melanomapatients who were treated with ipilimumab at 10 mg/kg/dose andsargramostim at 125 μg/m²/dose. Immune subsets were examined in PBMCfrom baseline, after cycle 1 and after cycle 2 of treatment with flowcytometry. As the number of patients who were alive is almost half ofall the patients, comparison of immune subsets were made betweenpatients with complete response, partial response and stable disease(responders, R, n=8) versus patients who had progressive disease(non-responder, NR, n=13)

Disclosed herein are the results that pre-treatment clinicalcharacteristics of patients where applicable, such as age, LDH levels,Eastern Cooperative Oncology group (ECOG) performance status, Gleasonscore of tumors, metastatic stages, prior treatments, and subsequenttherapies after leaving clinical trial did not relate with survival formCRPC patients and clinical responses for metastatic prostate cancerpatients.

It has been shown that the absolute lymphocyte counts after twoipilimumab treatments correlate significantly with clinical benefit andOS in a clinical trial for melanoma. Disclosed herein are the resultsthat for our clinical trials, the levels of absolute lymphocyte countsof patients at pre-treatment or after cycle 1 and cycle 2 of treatmentdid not relate with survival for mCRPC patients and clinical responsesfor metastatic prostate cancer patients.

Disclosed herein are the results that lower pre-treatment levels ofPD-1⁺ CD4 T_(eff) cells and higher pre-treatment levels of CTLA-4⁺ CD4 Tcells, were each significantly associated with better overall survivalfor mCRPC patients. The levels of these same immune subsets examinedafter cycle 1 and cycle 2 did not relate with survival. The levels ofthe parent subsets, total CD4 T cells, CD4 T_(eff) cells, and CD8 Tcells did not relate with survival at pre-treatment or after cycle 1 andcycle 2 of treatment.

Disclosed herein are the results that lower pre-treatment levels ofPD-L1⁺ CD4 T cells and PD-1⁺ CD4 T_(eff) cells were significantlyassociated with clinical responses for metastatic melanoma patients. Thelevels of PD-L1⁺ CD4 T cells and PD-1⁺ CD4 T_(eff) cells also relatewith survival after cycle 2 but not after cycle 1 of treatment. Thelevels of total CD4 T cells also relate with responses at pre-treatment,after cycle 1 and cycle 2 of treatment.

Disclosed herein are the results that mCRPC patients with poorersurvival have significantly higher pre-treatment levels of PD-1⁺ CD4T_(eff) cells compared to cancer-free controls, whereas patients withbetter survival have similar pre-treatment levels of PD-1⁺ CD4 T_(eff)cells compared to cancer-free controls.

Disclosed herein are the results that mCRPC patients with poorersurvival have similar or slightly higher pre-treatment levels of CTLA-4⁺CD4 T cells compared to cancer-free controls, whereas patients withbetter survival have significantly higher pre-treatment levels ofCTLA-4⁺ CD4 T cells compared to cancer-free controls.

Disclosed herein are the results that metastatic melanoma patients withprogressive disease have significantly higher pre-treatment levels ofPD-L1⁺ CD4 T cells and PD-1⁺ CD4 T_(eff) cells compared to cancer-freecontrols, whereas patients with clinical responses or stable diseasehave similar pre-treatment levels of PD-L1⁺ CD4 T cells and PD-1⁺ CD4T_(eff) cells compared to cancer-free controls.

Disclosed herein are the results that a proportion of these PD-1⁺ CD4T_(eff) cells express granzyme B without activation and IFNy uponPMA/ionomycin activation, which is consistent with an effector T cellphenotype.

Without wishing to be bound by theory, a potential explanation for theassociation of PD-1⁺ and PD-L1⁺ CD4 T cells with poorer survival is thatCTLA-4 blockade with ipilimumab treatment removes inhibition on T cellsthat express CTLA-4, but tolerance to tumor antigens maintained by PD-1⁺or PD-L1⁺ CD4 T cells persists and results in poorer survival. Inaddition it may also explain the increased effectiveness of combinedCTLA-4 and PD-1 blockade therapies (10), especially as CTLA-4 blockadealso increased the levels of PD-1⁺ CD4 effector T cells duringtreatment. It is expected that low PD-1 and/or low PD-L1 but high CTLA-4levels in CD4 T cells will be useful as biomarkers for improved survivalfollowing CTLA-4 blockade immunotherapy and it is expected that highPD-1 but low CTLA-4 levels will characterize patients suitable forcombination immunotherapy with anti-PD-1 and anti-CTLA-4immunotherapeutics.

The following examples illustrate embodiments of the disclosure.

EXAMPLE 1 Materials and Methods Clinical Trial

For mCRPC, the results for the lower dose levels up to 3 mg/kg/dose forthis phase 1b trial have been previously described (13). Briefly,patients had histologically proven metastatic castration resistantadenocarcinoma of the prostate with progression as defined by the PSAWorking Group Consensus Criteria (14). Patients had received no priorsteroids, chemotherapy or immunotherapy treatment. Patients receivedescalating doses of ipilimumab (Bristol-Myers Squibb) with a fixed doseof sargramostim (Sanofi). The initial design included dose escalation ofipilimumab from 0.5 mg/kg to 3 mg/kg (0.5, 1.5, and 3) every 4 weeks for4 doses (13). The study was subsequently modified to include 5 and 10mg/kg dose levels, as well as an expansion cohort of 6 patients at 3mg/kg/dose (cohort 5A) (Table 1). Sargramostim at 250 μg/m2/dose on days1-14 of 28 days cycles was administered subcutaneously and continueduntil disease progression or grade 3 or 4 treatment-related toxicity.

For mCRPC, the primary endpoint of safety was graded according toNational Cancer Institute (NCI) Common Terminology Criteria for AdverseEvents version 3.0. Dose-limiting toxicity (DLT) included grade 3 or 4treatment-related toxicity but excluded grade 3 immune-related adverseevents (unless ocular) that did not require the use of steroids.Exploratory endpoints included T-cell activation, objective tumorresponses (decrease in tumor size and/or lesions) as defined by ResponseEvaluation Criteria in Solid Tumors (RECIST) (15), and PSA declines of≥50% in PSA levels confirmed 4 weeks later defined by the PSA WorkingGroup Consensus Criteria. Progression is defined as a 50% rise in PSAabove the nadir or back to baseline, whichever is lower, on at least twoconsecutive measurements at least two weeks apart; or the appearance ofone or more new lesions occurring more than one month after theinitiation of therapy. Bone scans (and CT scans if abnormal) wererepeated every 12 weeks and at the time of PSA progression. Best PSAdecline was the maximum percentage (%) decline from initial PSA levelsbefore treatment. Overall survival (OS) was calculated from date offirst treatment to date of death (n=40) or censor date of trial on Oct.21, 2014 (n=2).

For metastatic melanoma, inclusion criteria were histologicallyconfirmed, surgically incurable or unresectable, Stage III or IVmetastatic melanoma. Patients must have had disease progressionfollowing 1 systemic therapy for metastatic disease, a minimum of 1measurable lesion according to irRC criteria, ECOG performance status of0-2, and LDH≤4× upper limit of normal (ULN). Patients had nouncontrolled brain metastasis, no history of autoimmune disease, and noprior immunotherapy treatments. Patients were treated with treated with4 courses of GM-CSF and ipilimumab administered every 3 weeks. GM-CSFwill be administered subcutaneously daily in a dose of 125 μg/m²beginning on day 1 to day 14 of each 21-day cycle and ipilimumab will beadministered intravenously in a dose of 10 mg/kg on day 1 of each cycle.After the initial 3 months (4 cycles) of treatment, GM-CSFadministration continued for 4 additional cycles on the same scheduleand dose without ipilimumab for 14 days every 21 days until month 6.Maintenance therapy began at month 6 and consisted of ipilimumab in thesame dose combined with 14 days of GM-CSF. Administration of thiscombination was repeated every 3 months for up to 2 years or untildisease progression, whichever occurred first.

For metastatic melanoma, primary end point is disease control rate at 24weeks. Secondary endpoints are assessment of immune activation, durationof disease control, overall survival, objective response rate using theimmune related Response Criteria (irRC) (11), time to objectiveresponse, duration of objective response (CR or PR), and safety of thecombination as defined by the NCI CTCAE criteria Version 4.0. Diseaseassessments will be performed using CT scans of the chest, abdomen, andpelvis and MRI scan of the brain at screening and every 3 months. OS wascalculated from date of first treatment to date of death (n=11) orcensor date of trial on Feb. 12, 2015 (n=10).

Flow Cytometry

Staining for flow cytometry was carried out on cryopreserved peripheralblood mononuclear cells (PBMC). In addition to study participants, PBMCwere also obtained from men undergoing prostate cancer screening withouta subsequent diagnosis of cancer (cancer-free male controls). Cellsurface staining was performed in FACS buffer for 30 min at 4° C.Intracellular FoxP3, CTLA-4, and Ki67 staining was performed using theFoxP3 fix/perm buffer set (Biolegend, Inc., 421403) according to themanufacturers' protocol. CD49b, granzyme B, and Lag-3 were stained usingthe intracellular fixation and permeabilization buffer set (eBioscience,Inc., 88-8824). Intracellular IFNy and IL-4 were stained suing the Foxp3staining buffer set (eBioscience, Inc., 00-5523) after PBMC wereactivated with 50 ng/ml PMA and 1 ionomycin for four hours at 37° C. inthe presence of 5 μg/ml of Brefeldin A for the last two hours ofactivation. The following anti-human antibodies were used: (A700)-CD3(Biolegend, Inc., 300324), (BV570)-CD4 (Biolegend, Inc., 300533),(PerCP/Cy5.5)-CD8 (Biolegend, Inc., 301032), (BV650)-CD25 (Biolegend,Inc., 302633), (FITC)-CD49b (Biolegend, Inc., 359306), (A647)-CD127(Biolegend, Inc., 351318), (PE)-CTLA-4 (Biolegend, Inc., 349906),(A488)-FoxP3 (Biolegend, Inc., 320112), (PE)-Granzyme B (BD Biosciences,561142), (BV650)-IFNy (Biolegend, Inc., 502537), (A647)-IL-4 (Biolegend,Inc., 500712), (APC)-Lag-3 (eBioscience, Inc., 3DS223H), (BV421)-PD-1(Biolegend, Inc., 329920), and (PE-Cy7)-PD-L1 (BD Biosciences, 558017).Stained cells were analyzed with an LSRII (BD Biosciences) flowcytometer. Data analysis was performed with Flowjo software (Treestar).Percentage (%) of positive cells was gated based on appropriate isotypecontrol. Absolute count for each immune subset is calculated bymultiplying the percentage of each subset with the preceding parentsubset and with the absolute lymphocyte count quantitated on the day ofblood drawn for that sample.

Statistical Analysis

Distributions of percentage of paired immune subsets at pre-treatment(week 0) were compared with cycle 1 (week 4 for mCRPC patients; week 3for metastatic melanoma patients) or with cycle 2 (week 8 for mCRPCpatients; week 6 for metastatic melanoma patients) using Wilcoxonmatched-pairs signed rank test using Prism (GraphPad) software. Thenumber of patients with PBMC at the various time points differed basedon availability.

Distributions of categorical patient characteristics such as ECOGstatus, Gleason score, prior radical prostatectomy, prior radiation,subsequent therapies, and clinical responses for mCRPC patients, sex,tumor stage, presence of immune adverse related events, prior therapy,and prior systemic therapy for metastatic melanoma patients werecompared using Fisher's exact test with Prism (Graphpad) software.

Distributions of continuous patient characteristics where applicable,such as age, baseline PSA levels, lactate dehydrogenase (LDH) levels,months on study, percentage or absolute counts of immune subsets betweenlong-term survivors (LTS) and short-term survivors (STS), and percentageor absolute counts of immune subsets between responders (R) andnon-responders (NR). Percentage of immune subsets between cancer-freemale controls and LTS or STS or R or NR were similarly compared usingMann-Whitney U-test.

Comparison of overall survival of cancer patients divided into twogroups based on cutoff levels of immune subsets were carried out byplotting Kaplan-Meier curves for each group and carrying out log-ranktest.

Statistical significance was declared based on alpha level of 0.05 withBonferroni correction to adjust for multiple testing as needed. Due tothe small sample size, all significant outcomes should be considered ashypothesis generating and confirmation with a larger sample size areneeded.

EXAMPLE 2 Patient Characteristics

TABLE 1 Patient Characteristics for mCRPC N = 42 Median (range) Age(years) 72.5 (52-82) ECOG PS (n) 0 31 1 11 Gleason score ≤6  8  7 11 ≥821 Baseline PSA (ng/mL) 37.5 (6.7-435) LDH (U/L)  172 (136-557) Alkalinephosphatase (U/L)   92 (28-1725) Metastases Bone 25 Soft tissue  5 Both12

TABLE 2 Patient Characteristics for metastatic melanoma N = 22Percentage (range) Age, median (range), years   65 (41-85) Sex Men 12(54.5) Women 10 (45.5) Metastatic Stage Unresectable III  1 (4.55) Mia 1 (4.55) Mlb  5 (22.7) Mlc 15 (68.2) Sites of Metastasis Lymph Nodes 14(63.6) Lung 14 (63.6) Liver 10 (45.5) Bone  7 (31.8) Subcutaneous tissue 6 (27.3) CNS  4 (18.2) Skin  3 (13.6) Adrenal  3 (13.6) Intestine  1(4.55) Spleen  1 (4.55) Retroperitoneum  1 (4.55) Received priorsystemic therapy No 15 (68.2) Yes  7 (31.8) Prior Therapy Radiation  9(40.9) Systemic, Chemo  4 (18.2) Adjuvant  3 (13.6) Systemic, Non-Chemo2 (9.1) Localized 1 (4.5)

EXAMPLE 3 Clinical Outcomes

A waterfall plot of nadir PSA values (FIG. 2A) demonstrates that 23 outof 42 patients (54%) had some decline in PSA. Five of 42 patients(11.9%) experienced a 50% or greater decline in PSA (Table 1). Themedian time to PSA nadir was 5.9 weeks (range 1.9-39.1 weeks) forpatients with any PSA decline and 15.9 weeks (range 11.9-39.1 weeks) forpatients with at least a 50% PSA decline (FIG. 2B). Objective tumorresponse and at least a 50% PSA decline was not observed in cohortstreated at less than 3 mg/kg/dose level. Three out of 12 patientstreated at 3 mg/kg/dose experienced at least a 50% PSA decline, of whomtwo had objective responses with regression of liver metastasis in onepatient and bone metastasis in another (cohort 5). One patient in theexpansion cohort at 3mg/kg experienced a 49% decline (cohort 5A). In thecohort treated at 5 mg/kg/dose, none of 6 patients demonstrated at leasta 50% PSA decline or objective tumor response. Of the 6 patients treatedat the 10 mg/kg, two had at least a 50% PSA decline. No accompanyingobjective tumor response was seen.

As of censor date of the trial, all patients had come off study. Onepatient came off treatment by patient's choice. 13 patients came offtreatment for PSA progression prior to the first set of scans, 16patients came off treatment with PSA progression following the first setof scans, 6 patients came off treatment for tumor progression by scans,and 6 patients came off treatment for immune-related adverse toxicities.However, two patients from the 3 mg/kg/dose group whom came offtreatment due to immune-related toxicities demonstrated durableresponses with their PSA levels remaining less than 50% of theirpre-treatment levels for 19 and 85 months after being off treatmentwithout any new treatment (FIG. 2C). One patient from the 5 mg/kg/dosegroup came off treatment due to an initial disease progression, but adelayed response was observed with his PSA decline attaining 50% at 7months without any new treatment.

As this is a Phase 1b study, survival analysis was not specified in theprotocol. As immunotherapies can induce improvements in overall survivalwithout conventional progression, survival analysis was carried outpost-hoc. The median OS for all the patients (n=42) is 23.6 months (95%confidence limits {CI}={16.2, 39.3}) (FIG. 2D).

TABLE 3 Clinical Responses for mCRPC >50% PSA Median Response Overall(Best Objective TTP^(c) Survival Dose Level^(a) decline %) Response^(b)(months) (months) 1 0/3 0/3 25 (0.5 mg/kg × 4) 2 0/7 0/7 26 (1.5 mg/kg ×1, 0.5 mg/kg × 3) 3 0/5 0/5 28 (1.5 mg/kg × 4) 4 0/3 0/3 12 (3 mg/kg ×1, 1.5 mg/kg × 3) 5 3/6 2/6 20, 25.75, 56 (3 mg/kg × 4) (79, 95, 97)89.25 6 0/6 0/6 13 (5 mg/kg × 4) 7 2/6 0/6 9.75, 18 19 (10 mg/kg × 4)(50, 80) 5A 0/6 0/6 20 (3 mg/kg × 4) Cumulative 5/42  2/42  20 (median)  23.6 ^(a)Dosage of ipilimumab and the number of doses are given inbrackets ( ); ^(b)Objective tumor response defined by RECIST; ^(c)TTP istime to progression calculated from the time of initial response.

Clinical outcomes for metastatic melanoma: Out of 21 patients who hadreceived at least 2 cycles of treatment, 1 patient had CR, 6 patientshad PR, 1 patient had SD, and 13 patients had PD. The median OS for allthe treated patients (n=22) was 21.4 months (FIG. 3A). Overall survivalassociated significantly with clinical responses (CR+PR) plus SD (FIG.3B) (p-value=0.005).

EXAMPLE 4 Toxicity

Consistent with the known toxicity profile of ipilimumab, toxicity wasprimarily immune in nature.

TABLE 4 Adverse events for mCRPC Dose Dose-Limiting Level All adverseevents^(a) Toxicities 1 1/3 0/3 grade 1: nausea (1) 2 1/7 1/7 grade 3:CVA (1) grade 3: CVA 3 2/5 1/5 grade 3: fatigue (1), rash (1) grade 3:rash requiring steroids 4 0/3 0/3 5 4/6 1/6 grade 2: muscle spasms (1)grade 4: CVA grade 3: angina (1), temporal arteritis (1), diarrhea (1),panhypopituitarism (1) grade 4: CVA (1) 6 5/6 1/6 grade 1: fatigue (1),muscle spasms (1), grade 5: PE diarrhea (2) grade 2: wheezing (1), hotflashes (1), fatigue (1), pruritus (2), rash (3) grade 3: fatigue (1),atrial fibrillation (1) grade 5: PE (1) 7 6/6 1/6 grade 1: diarrhea (1),rash (1) grade 3: rash grade 2: vomiting (1), dehydration (1), requiringsteroids pruritus (3), fatigue (1), erythema (1), adrenal insufficiency(2) grade 3: fatigue (2), diarrhea (2), rash (3) grade 4: elevatedtroponin (1) 5A^(b) 4/4 1/4 grade 1: increased LFT (1) grade 3: diarrheagrade 2: adrenal insufficiency (1), requiring steroids pneumonitis (1)grade 3: atrial fibrillation (1), DVT (1), diarrhea (1) grade 4: fatigue(1) ^(a) Immune-related adverse events are in bold. The fraction ofpatients with any adverse event is presented per cohort. The number ofpatients with each adverse event is listed in brackets ( ). As a patientmight have experienced more than one adverse event, the sum of alladverse events may be greater than the number of patients in eachcohort; ^(b)Information for adverse events was available only for fourout of six patients in this cohort; CVA, cerebrovascular accidents; DVT,deep venous thrombosis; LFT, liver function test; PE, pulmonaryembolism.

TABLE 5 Adverse events for metastatic melanoma % % Grades GradesToxicity 1-2 3-4 Fatigue 50 6.25 Injection Site Reaction 43.75 6.25Infusion Reaction 12.5 6.25 Fever 12.5 0 Flu-like symptoms 6.25 0Irritability 6.25 0 Flushing 6.25 0 Gastrointestinal 50 37.5 Diarrhea18.75 12.5 Nausea 31.25 0 Colitis 0 18.75 Colon Perforation 0 6.25 Skinand Subcutaneous 81.25 6.25 Rash 31.25 6.25 Pruritis 31.25 0 Urticaria12.5 0 Hyperhidrosis 6.25 0 Anorexia 37.5 6.25 Investigational 25 6.25Weight loss 12.5 0 Decreased ACTH 0 6.25 Increased AST 6.25 0 IncreasedALT 6.25 0 Nervous System 25 0 Headache 12.5 0 Tremor 6.25 0 Dysgeusia6.25 0 Cardiac 12.5 6.25 Palpitations 6.25 0 Atrial fibrillation 0 6.25Pericarditis 6.25 0 Endocrine 6.25 6.25 Hyperthyroidism 6.25 6.25Musculoskeletal 12.5 0 Myalgias 6.25 0 Arthalgias 6.25 0 Infection 6.250 Phayrngitis 6.25 0

EXAMPLE 5 Patients' Baseline Characteristics did not Relate withSurvival for mCRPC Patients or Clinical Responses for MetastaticMelanoma Patients

For mCRPC patients, age, baseline PSA levels, LDH levels, months onstudy, did not relate with OS (p-values=0.193, 0.311, 0.277, and 0.100respectively). The number of patients with ECOG status of 0 or 1,Gleason scores grouped as 3 to 6, or 7 to 9, prior radicalprostatectomy, and prior radiation, were not significantly differentbetween the two groups (p-values=1.00, 0.90, 1.00, and 0.67respectively). The number of patients with clinical responses asdescribed above and the number of patients who went on to subsequenttherapies also did not correlate with OS (p-values=0.16 and 0.38respectively) (FIG. 11).

For metastatic melanoma, age, sex, tumor stage, prior therapy, immuneadverse events, and prior systemic therapy did not relate significantlywith either responders (R) or non-responders (NR) (p-values=0.23, 0.66,0.71, 0.67, 0.39, and 0.40 respectively) (FIG. 12).

EXAMPLE 6 Absolute Lymphocyte Counts did not Associate with Survival formCRPC Patients or Clinical Responses for Metastatic Melanoma Patients

For mCRPC patients, the absolute lymphocyte counts were significantlyhigher compared to pre-treatment levels after cycle 1 but not aftercycle 2 of treatment (p-values=0.002 and 0.119 respectively) (FIG. 7A).The distribution of the absolute lymphocyte counts however did notdiffer between LTS and STS at pre-treatment (p-value=0.201), after cycle1 (p-value=0.670), and after cycle 2 of treatment (p-value=0.779) (FIG.7B).

For metastatic melanoma patients, the absolute lymphocyte counts weresignificantly higher compared to pre-treatment levels after cycle 1 andafter cycle 2 of treatment (p-values=0.0002 and <0.0001 respectively)(FIG. 8A). The distribution of the absolute lymphocyte counts howeverdid not differ between R and NR at pre-treatment (p-value=0.753), aftercycle 1 (p-value=0.983), and after cycle 2 of treatment (p-value=0.126)(FIG. 8B).

EXAMPLE 7 Relationship of CD4 Teff Cells, Total CD4 T Cells and CD8 TCells with Survival or Clinical Responses

For mCRPC, the percentages of total lymphocytes and absolute counts ofCD4 T_(eff) cells (CD4⁺CD3⁺FoxP3⁻) were significantly higher after onecycle of treatment (FIGS. 7C and E). However, for CD8 T cells, only theabsolute counts and not the percentage of total lymphocytes, weresignificantly higher after one cycle of treatment (FIGS. 7G and I). Thisdifference could be due to the higher levels of absolute lymphocytecounts after one cycle of treatment as described above.

For mCRPC patients, The distribution of the percentages of totallymphocytes and absolute counts of CD4 T_(eff) cells did not differbetween LTS and STS at pre-treatment (p-values=0.263 and 0.841respectively), after cycle 1 (p-values=0.805 and 0.745 respectively),and after cycle 2 of treatment (p-values=0.920 and 0.845 respectively)(FIG. 7D, F). The percentages of total lymphocytes and absolute countsof CD8 T cells also did not relate with survival at pre-treatment(p-values=0.461 and 0.304 respectively), after cycle 1 (p-values=0.555and 0.670 respectively), and after cycle 2 of treatment (p-values=0.671and 0.835 respectively) (FIG. 7H, J).

For metastatic melanoma patients, the percentages of total lymphocytesand absolute counts of total CD4 T cells (CD8⁻CD3⁺) were significantlyhigher after 1 and 2 cycles of treatment (FIGS. 8C and E). Thepercentages of total lymphocytes and absolute counts of total CD8 Tcells (CD8⁺CD3⁺) were significantly higher after 1 and 2 cycles oftreatment (FIGS. 8G and I).

The distribution of the percentages of total lymphocytes but not theabsolute count of CD4 T cells did not differ between R and NR atpre-treatment (p-values=0.025 and 0.064 respectively), was significantlydifferent for percentages but not absolute counts after cycle 1(p-values=0.008 and 0.076 respectively), and was significantly differentfor both percentages and absolute counts after cycle 2 of treatment(p-values=0.033 and 0.026, respectively) (FIG. 8D, F). The percentagesof total lymphocytes and absolute counts of CD8 T cells did not relatewith clinical responses at pre-treatment (p-values=0.053 and 0.886). Thepercentages of total lymphocytes and absolute counts of CD8 T cellsafter cycle 1 did not correlate with clinical responses (p-values=0.053and 0.074, respectively). The percentages of total lymphocyte but notthe absolute counts of CD8 T cells related significantly with clinicalresponses after cycle 2 of treatment (p-values=0.035 and 0.372,respectively) (FIG. 8H, J).

EXAMPLE 8 Lower Pre-Treatment Levels of PD-1⁺ CD4 Effector T CellsAssociate with Longer Survival for mCRPC Patients and with ClinicalResponses for Metastatic Melanoma Patients

For mCRPC patients, the percentages of CD4 T_(e) _(ff) cells thatexpress surface PD-1 were significantly higher after cycle 1(p-value=0.0001) and continued to be significantly higher after cycle 2compared to pre-treatment levels (p-value=0.0002) (FIG. 4A, B, D). Theabsolute counts of PD-1+ CD4 T_(e) _(ff) cells were also significantlyhigher after both cycles (p-values=0.0001 and 0.0002 respectively). Thepercentages of CD8 T cells that express PD-1 were also significantlyhigher from pre-treatment levels after cycle 1 (p-value=0.004) and aftercycle 2 of treatment (p-value=0.005) (FIG. 4A, C, E). The absolutecounts of PD-1⁺ CD8 T cells were also significantly higher after bothcycles (p-values=0.005 and 0.022 respectively).

For mCRPC patients, the distribution of the percentages of surface PD-1+on CD4 T_(e) _(ff) cells and absolute counts of PD-1⁺ CD4 T_(e) _(ff) atpre-treatment was significantly lower in LTS compared to STS(p-values=0.0007 and 0.003 respectively) (FIG. 4F, H). After treatment,the distribution of the percentages of surface PD-1⁺ on CD4 T_(e) _(ff)cells and absolute counts of PD-1⁺ CD4 T_(e) _(ff) were notsignificantly different between STS or LTS after cycle 1 (p-values=0.055and 0.090 respectively) and after cycle 2 (p-values=0.054 and 0.150respectively) (FIG. 4F, H). The distributions of the percentages andabsolute counts of surface PD-1⁺ CD8 T cells between STS and LTS did notdiffer at pre-treatment (p-value=0.246 and >0.999 respectively) or atany time point after treatment (FIG. 4A, G, I).

For metastatic melanoma patients, the percentages of CD4 T_(e) _(ff)cells that express surface PD-1 similarly increased after cycle 1 andcycle 2 (p-values=<0.0001 for both). The absolute counts of PD-1⁺ CD4T_(e) _(ff) cells were similarly higher after both cycles(p-values=<0.0001 and 0.0001 respectively). The percentages of CD8 Tcells that express PD-1 were also significantly higher frompre-treatment levels after cycle 1 (p-value=0.001) and after cycle 2 oftreatment (p-value=0.003). The absolute counts of PD-1⁺ CD8 T cells werealso significantly higher after both cycles (p-values=<0.0001 for both)(FIG. 14).

For metastatic melanoma patients, the distribution of the percentages ofsurface PD-1+ on CD4 T_(e) _(ff) cells but not the absolute counts ofPD-1⁺ CD4 T_(e) _(ff) at pre-treatment was significantly lower in Rcompared to NR (p-values=0.017 and 0.887, respectively). Thedistribution of the percentages of surface PD-1+ on CD4 T_(e) _(ff)cells but not the absolute counts of PD-1⁺ CD4 T_(e) _(ff) after cycle 2was significantly lower in R compared to NR (p-values=0.040 and 0.946respectively). The distributions of the percentages and absolute countsof surface PD-1⁺ CD8 T cells between R and NR did not differ atpre-treatment (p-values=0.453 and 0.744 respectively) or at any timepoint after treatment (cycle 1 percentage and absolute countp-values=0.543 and 0.441; cycle 2 percentage and absolute countp-values=0.229 and 0.118).

EXAMPLE 9 High Pre-Treatment Levels of CLTA-4⁺ CD4 T Cells Associatewith Improved Survival

For mCRPC patients, the percentages of CTLA-4⁺ of CD4 T cells increasedsignificantly after cycle 1 (p-value=0.0078) and after cycle 2(p-value=0.016) of treatment (FIGS. 5A, and B). The absolute counts ofCTLA-4⁺ CD4 T cells also increased significantly after cycle 1(p-value=0.0078) and after cycle 2 (p-value=0.016) of treatment (FIG.5D). The percentages of CTLA-4⁺ of CD8 T cells increased significantlyafter cycle 1 (p-value=0.0078) and after cycle 2 (p-value=0.016) oftreatment (FIGS. 5A, and C). The absolute counts of CTLA-4⁺ CD8 T cellsalso increased significantly after cycle 1 (p-value=0.0078) and aftercycle 2 (p-value=0.016) of treatment (FIG. 5E).

For mCRPC patients, higher pre-treatment percentages of CTLA-4⁺ of CD4 Tcells at pre-treatment related with long-term survivors (p-value=0.030)but not after cycle 1 or cycle 2 of treatment (p-values=0.524 and >0.999respectively) (FIG. 5F). The pre-treatment absolute CTLA-4⁺ CD4 T cellcount was also significantly different between LTS and STS atpre-treatment (p-value=0.041 1) (FIG. 5H) but not after cycle 1 or cycle2 of treatment (p-values=0.1 11 for both). The distribution of thepercentages of CTLA-4⁺ of CD8 T cells at pre-treatment or after cycles 1or 2 did not relate with long-term survivors (p-values=0.788, 0.508, and0.683 respectively) (FIG. 5G). The pre-treatment absolute CTLA-4⁺ CD8 Tcell count was also not significantly different between LTS and STS atpre-treatment or after cycles 1 or 2 (p-values=>0.999, 0.83, and >0.999respectively) (FIG. 51).

EXAMPLE 10 Lower Pre-Treatment Levels of PD-L1⁺ CD4 effector T CellsAssociate with Clinical Responses for Metastatic Melanoma Patients

For metastatic melanoma patients, the percentages of PD-L1⁺ of CD4 Tcells did not increased significantly after cycle 1 (p-value=0.058) butincreased significantly after cycle 2 (p-value=0.004) of treatment(FIGS. 6A, and B). The absolute counts of PD-L1⁺ CD4 T cells increasedsignificantly after cycle 1 (p-value=<0.0001) and after cycle 2(p-value=0.0004) of treatment (FIG. 6D). The percentages of PD-L1⁺ ofCD8 T cells did not increased significantly after cycle 1(p-value=0.202) and after cycle 2 (p-value=0.562) of treatment (FIGS.6A, and C). The absolute counts of PD-L1⁺ CD8 T cells increasedsignificantly after cycle 1 (p-value=0.012) and after cycle 2(p-value=0.001) of treatment (FIG. 6E).

For metastatic melanoma patients, lower pre-treatment percentages ofPD-L1⁺ of CD4 T cells at pre-treatment and after cycle 2 related withclinical responses (p-values=0.006 and 0.007 respectively) but not aftercycle 1 of treatment (p-value=0.087) (FIG. 6F). The pre-treatmentabsolute PD-L1⁺ CD4 T cell count was not significantly different betweenR and NR at pre-treatment (p-value=0.268) (FIG. 6H) or after cycle 1 orcycle 2 of treatment (p-values=0.065 and 0.111 respectively). Thedistribution of the percentages of PD-L1⁺ of CD8 T cells atpre-treatment or after cycles 1 or 2 did not relate with clinicalresponses (p-values=0.727, 0.962, and 0.643 respectively) (FIG. 6G). Thepre-treatment absolute PD-L1⁺ CD8 T cell count was also notsignificantly different between R and NR at pre-treatment or aftercycles 1 or 2 (p-values=0.244, 0.399, and 0.181 respectively) (FIG. 61).

EXAMPLE 11 Comparison with Cancer-Free Controls

mCRPC patients with poorer survival have significantly higherpre-treatment levels of PD-1⁺ CD4 T_(e) _(ff) cells compared tocancer-free controls (p-value=0.001), whereas patients with bettersurvival have similar pre-treatment levels of PD-1⁺ CD4 T_(e) _(ff)cells compared to cancer-free controls (FIG. 9A).

mCRPC patients with poorer survival have similar or slightly higherpre-treatment levels of CTLA-4⁺ CD4 T cells compared to cancer-freecontrols, whereas patients with better survival have significantlyhigher pre-treatment levels of CTLA-4⁺ CD4 T cells compared tocancer-free controls (p-value=0.001) (FIG. 9B).

Metastatic melanoma patients with progressive disease have significantlyhigher pre-treatment levels of PD-1⁺ CD4 T_(e) _(ff) cells compared tocancer-free controls (p-value=0.0002), whereas patients with clinicalresponses or stable disease have similar pre-treatment levels of PD-L1⁺CD4 T cells compared to cancer-free controls (FIG. 9C).

Metastatic melanoma patients with progressive disease have significantlyhigher pre-treatment levels of PD-L1⁺ CD4 T cells compared tocancer-free controls (p-value=0.047), whereas patients with clinicalresponses or stable disease have similar pre-treatment levels of PD-L1⁺CD4 T cells compared to cancer-free controls (FIG. 9D).

EXAMPLE 12 Summary of Significance Values Percentages of Immune Subsets

TABLE 6 Comparison of pre-treatment T cell subsets LTS (n = 8) STS (n =12) mCRPC patients Median^(a) (Range^(b)) Median^(a) (Range^(b))p-value^(c) Total CD4 T cells (CD4⁺CD3⁺) 41.0 (32.8-59.6) 48.3(33.6-71.8) 0.203 CD4 T_(eff) cells (CD4⁺CD3⁺FoxP3⁻) 38.9 (30.1-56.2)45.2 (30.4-66.8) 0.263

10.1 (5.4-14.5) 22.0 (12.8-42.3) 0.0007 Total CD8 T cells (CD4⁻CD3⁺)24.5 (5.0-42.2) 18.3 (6.7-50.7) 0.461 PD-1⁺CD4⁻CD3⁺ 15.0 (5.3-28.0) 22.2(8.4-33.0) 0.246 LTS (n = 6) STS (n = 6) mCRPC patients Median^(a)(Range^(b)) Median^(a) (Range^(b)) p-value^(c) Total CD4 T cells(CD8⁻CD3⁺⁾ 43.0 (35.2-58.9) 51.7 (40.6-61.9) 0.305

18.7 (14.2-21.6) 13.7 (9.4-19.9) 0.030 Total CD8 T cells (CD8⁺CD3⁺) 10.8(4.2-25.0) 12.9 (5.4-24.5) 0.675 CTLA-4 ⁺CD8⁺CD3⁺  6.2 (2.1-12.2)  5.5(3.7-12.0) 0.788 R (n = 8) NR (n = 13) Metastatic melanoma patientsMedian^(a) (Range^(b)) Median^(a) (Range^(b)) p-value^(c) CD4 Teff cells(CD4⁺CD3⁺FoxP3⁻) 46.9 (24.3-58.9) 38.2 (19.1-53.6) 0.104

14.6 (11.5-33.9) 30.2 (15.4-43.8) 0.017 Total CD8 T cells (CD4⁻CD³⁺)18.9 (13.3-39.6) 29.8 (16.4-64.4) 0.157 PD-1⁺CD4⁻CD3⁺ 31.2 (19.2-53.4)28.5 (9.3-56.9) 0.453 R (n = 8) NR (n = 13) Metastatic melanoma patientsMedian^(a) (Range^(b)) Median^(a) (Range^(b)) p-value^(c) Total CD4 Tcells (CD8⁻CD3⁺⁾ 56.1 (37.4-69.1) 43.0 (20.4-61.6) 0.025

15.1 (9.2-24.7) 24.4 (18.1-38.1) 0.006 Total CD8 T cells (CD8⁺CD3⁺⁾ 13.7(6.1-27.3) 23.7 (4.5-63.4) 0.053 PD-L1⁺CD8⁺CD3⁺  7.0 (3.9-19.4)  6.7(3.8-10.3) 0.727 ^(a)Median values of parent gates are % of totallymphocytes and median values of immune checkpoint markers are % of therespective parent gate; ^(b)Values in brackets ( ) are range of eachdata set; ^(c)Mann-Whitney test; Bold-faced characters highlight p-value≤ 0.05; LTS, long-term survivors; STS, short-term survivors; R, clinicalresponders; NR, non-responders.

Absolute Counts of Immune Subsets

TABLE 7 Comparison of pre-treatment absolute counts of T cell subsetsLTS (n = 8) STS (n = 12) mCRPC patients Median^(a) (Range^(b))Median^(a) (Range^(b)) p-value^(c) Total CD4 T cells (CD4⁺CD3⁺) 0.80(0.48-0.99) 0.64 (0.33-1.15) 0.841 CD4 T eff cells (CD4⁺CD3⁺FoxP3⁻) 0.74(0.45-0.91) 0.61 (0.27-1.07) 0.841

0.07 (0.04-0.10) 0.12 (0.07-0.30) 0.003 Total CD8 T cells (CD4⁻CD3⁺)0.44 (0.05-0.79) 0.22 (0.09-1.17) 0.304 PD-1 ⁺CD4⁻CD3⁺ 0.05 (0.01-0.10)0.03 (0.02-0.37) >0.999  LTS (n = 6) STS (n = 6) mCRPC patientsMedian^(a) (Range^(b)) Median³ (Range^(b)) p-value^(c) Total CD4 T cells(CD8⁻CD3⁺) 1.02 (0.62-1.25) 0.65 (0.60-1.03) 0.387

0.19 (0.09-0.23) 0.09 (0.06-0.20) 0.041 Total CD8 T cells (CD8⁺CD3⁺)0.24 (0.03-0.67) 0.19 (0.07-0.32) 0.788 CTLA-4 ⁺CD8⁺CD3⁺ 0.01(0.003-0.05) 0.009 (0.005-0.04) >0.999  R (n = 8) NR (n = 13) Metastaticmelanoma patients Median^(a) (Range^(b)) Median³ (Range^(b)) p-value^(c)CD4 T_(eff) cells (CD4⁺CD3⁺FoxP3⁻) 0.80 (0.13-1.31) 0.40 (0.12-1.08)0.064 0.12 (0.06-0.25) 0.11 (0.04-0.31) 0.886

0.35 (0.10-0.75) 0.26 (0.15-1.22) 0.744 Total CD8 T cells (CD4⁻CD3⁺)0.10 (0.03-0.20) 0.09 (0.03-27) 0.744 PD-1 ⁺CD4⁻CD3⁺ R (n = 8) NR (n =13) Metastatic melanoma patients Median^(a) (Range^(b)) Median^(a)(Range^(b)) p-value^(c) Total CD4 T cells (CD8⁻CD3⁺) 1.01 (0.20-1.61)0.46 (0.12-1.17) 0.064

0.11 (0.03-0.17) 0.12 (0.05-0.38) 0.268 Total CD8 T cells (CD8⁺CD3⁺)0.20 (0.03-0.52) 0.19 (0.06-1.21) 0.886 PD-L1 ⁺CD8⁺CD3⁺ 0.02(0.008-0.06) 0.01 (0.002-0.04) 0.244 ^(a)Median absolute counts of Tcell subsets; ^(b)Values in brackets ( ) are range of each data set;^(c)Mann-Whitney test; Bold-faced characters highlight p-value ≤ 0.05;LTS, long-term survivors; STS, short-term survivors; R, clinicalresponders; NR, non-responders.

Comparison with Cancer-Free Controls

TABLE 8 Comparison of pre-treatment T cell subsets between cancer-freecontrols and cancer patients mCRPC 

Groups

 

PD-1⁺ CD4 ¾ cells Cancer-free controls LTS week 0 STS week 0 11.7(6.7-14.3) 10.1 (5.4-14.5) 22.2 (8.4-33.0)

PD-1⁺ CD8 T cells Cancer-free controls LTS week 0 STS week 0 13.7(8.7-27.5) 15.0 (5.3-28.0) 22.2 (8.4-33.0)

mCRPC 

Groups

 

CTLA-4⁺ CD4 T cells Cancer-free controls LTS week 0 STS week 0  8.8(6.1-13.4) 18.7 (14.2-21.6) 13.7 (9.4-19.9)

CTLA-4⁺ CD8 T cells Cancer-free controls LTS week 0 STS week 0  3.1(3.0-10.6)  6.2 (2.1-12.2)  5.5 (3.7-12.0)

Metastatic melanoma

Groups

 

PD-1 +CD4 Teff cells Cancer-free controls R week 0 NR week 0 13.3(11.3-21.1) 14.6 (11.5-33.9) 30.2 (15.4-43.8)

PD-1 CD8 T cells Cancer-free controls R week 0 NR week 0 15.6 (9.2-26.8)31.2 (19.2-53.4) 28.5 (9.3-56.9)

Metastatic melanoma

Groups

 

PD-L1⁺ CD4 T cells Cancer-free controls R week 0 NR week 0 11.1(3.6-32.5) 15.1 (9.2-24.7) 24.4 (18.1-38.1)

PD-L1⁺ CD8 T cells Cancer-free controls R week 0 NR week 0  2.9(1.9-4.6)  5.0 (2.8-14.6)  4.6 (2.7-6.0)

^(a)cancer-free controls, n = 7, LTS, n = 8; STS, n = 12;^(b)cancer-free controls, n = 7, LTS, n = 6, STS, n - 6; ^(c)cancer-freecontrols, n = 8, R, n = 8, NR, n = 13; ^(d)Median values of immunecheckpoint markers are % of the respective parent gate; ^(e)Values inbrackets ( ) are range of each data set; ^(f)Mann-Whitney test;Bold-faced characters highlight p-value ≤ 0.05; LTS, long-termsurvivors; STS, short-term survivors; R, clinical responders; NR,non-responders.

EXAMPLE 13 Establishing Optimal Cutoff Levels of Immune Subsets withKaplan-Meier Plots and Log-Rank Test

Overall survival of mCRPC patients with ≤21% of PD-1⁺ of CD4 T_(eff)cells was significantly different from overall survival of patientswith >21% of PD-1⁺ of CD4 T_(e) _(ff) cells (p-value=0.0027, FIG. 10).Overall survival of mCRPC patients with ≤106 cells/μï of PD-1 ⁺CD4 T_(e)_(ff) cells was significantly different from overall survival ofpatients with >106 cells/μï of PD-1⁺CD4 T_(ef) _(f) cells(p-value=0.026, FIG. 10).

Overall survival of mCRPC patients with >15.6% of CTLA-4⁺ of CD4 T cellswas significantly different from overall survival of patients with≤15.6% of CTLA-4⁺ of CD4 T cells (p-value=0.012, FIG. 10). Overallsurvival of mCRPC patients with >99 cells/μï of CTLA-4⁺CD4 T cells wassignificantly different from overall survival of patients with ≤99cells/μï of CTLA-4 ⁺CD4 T cells (p-value=0.012, FIG. 10).

Overall survival of metastatic melanoma patients with <23.5% of PD-L1⁺of CD4 T cells was significantly different from overall survival ofpatients with >23.5% of PD-L1⁺ of CD4 T cells (p-value=0.027, FIG. 10).

EXAMPLE 14 Intracellular Cytokine Expression of PD-1⁺ CD4 T_(eff) Cells

Cytokine expression patterns of PD-1⁺ and PD-1⁻ CD4 and CD8 T cells ofpre-treatment PBMC from mCRPC patients were compared to PBMC fromhealthy donors (FIG. 11). Freshly thawed PBMC were stimulated with PMAand ionomycin for 4 hours in culture at 37° C., and intracellularlystained for IFNy and 11-4. About 40% of PD-1⁺ CD4 T cells expressed IFNycompared to 5% of PD-1⁻ CD4 T cells. On the other hand, almost 90% ofPD-1⁺ CD8 T cells expressed IFNy, compared to 30 to 50% of PD-1⁻ CD8 Tcells. The ratio of PD-1⁺ to PD-1⁻ T cells expressing IFNy issignificantly greater for CD4 than for CD8 T cells. More PD-1⁺ than PD-ΓCD4 T cells expressed IL-4 although the levels were low. CD8 T cells didnot express IL-4. The percentages of IFNy and IL-4-expressing PD-1⁺ andPD-Γ CD4 T cells from cancer patients were comparable to healthy donors.

Unstimulated pre-treatment PBMC from mCRPC patients and unstimulatedPBMC from healthy donors were also stained for surface CD49b and Lag-3,and for intracellular granzyme B (FIG. 11). More PD-1⁺ CD4 T cellsexpressed CD49b, Lag-3, and granzyme B than PD-Γ CD4 T cells. Thepercentages of Lag-3 expression were similar for cancer patients andhealthy donors. However, the percentages of granzyme B andCD49b-expressing PD-1⁺ CD4 T cells were higher in cancer patients thanin healthy donors. Granzyme B-expressing PD-Γ CD4 T cells were alsogreater in cancer patients than in healthy donors, whereasCD49b-expressing PD-γ CD4 were similar for cancer patients and healthydonors. CD8 T cells displayed a different pattern of expression ofgranzyme B compared to CD4 T cells. Similar percentages of PD-1⁺ andPD-γ CD8 T cells from cancer patients expressed granzyme B, and bothlevels were higher than those from healthy donors. More PD-1⁺ than PD-ΓCD8 T cells from healthy donors expressed granzyme B. Expressions ofLag-3 and CD49b were low and did not differ for PD-1⁺ and PD-γ CD8 Tcells, or for cancer patients and healthy donors.

Each of the references cited below is incorporated by reference hereinin its entirety, or in relevant part, as would be apparent from context.The references are cited throughout this disclosure using superscriptednumbers corresponding to the following numbered reference list.

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11. Hoos A. Endpoints for Immunotherapy Studies: Design and RegulatoryImplications; Proposal of a Clinical Development Paradigm for CancerImmunotherapy. 2008.

The disclosed subject matter has been described with reference tovarious specific and preferred embodiments and techniques. It should beunderstood, however, that many variations and modifications may be madewhile remaining within the spirit and scope of the disclosed subjectmatter.

1.-32. (canceled)
 33. A method of determining whether cancer isamendable to treatment with an anti-CTLA-4 antibody product, the methodcomprising: (a) isolating a population of peripheral blood mononuclearcells (PBMCs) from a subject having cancer and a cancer-free subject;(b) quantifying the percentages of total CD4+ T cells expressing PD-1,PD-L1, and/or CTLA-4 in each of the populations; (c) comparing thepercentages of total CD4+ T cells expressing PD-1, PD-L1, and/or CTLA-4in the population of PBMCs from the subject having cancer and thepercentages of total CD4+ T cells expressing PD-1, PD-L1, and/or CTLA-4in the population of PBMCs from the cancer-free subject; and (d)determining the subject having cancer is amenable to treatment with ananti-CTLA-4 antibody product if: (1) the percentage of total CD4+ Tcells expressing PD-1 in the population of PBMCs from the subject havingcancer is lower than the percentage of total CD4+ T cells expressingPD-1 in the population of PBMCs from the cancer-free subject; or (2) thepercentage of total CD4+ T cells expressing PD-L1 in the population ofPBMCs from the subject having cancer is lower than the percentage oftotal CD4+ T cells expressing PD-L1 in the population of PBMCs from thecancer-free subject; or (3) the percentage of total CD4+ T cellsexpressing CTLA-4 in the population of PBMCs from the subject havingcancer is higher than the percentage of total CD4+ T cells expressingCTLA-4 in the population of PBMCs from the cancer-free subject.
 34. Themethod of claim 33, wherein the anti-CTLA-4 antibody product is selectedfrom the group consisting of an anti-CTLA-4 antibody or fragmentthereof, an anti-CTLA-4 chimeric antibody, an anti-CTLA-4 CDR-graftedantibody, an anti-CTLA-4 single-chain antibody, an anti-CTLA-4single-chain variable fragment, an anti-CTLA-4 Fab antibody fragment, ananti-CTLA-4 Fab′ antibody fragment, an anti-CTLA-4 F(ab′)2 antibodyfragment, an anti-CTLA-4 bi-body, an anti-CTLA-4 tri-body, ananti-CTLA-4 diabody, and an anti-CTLA-4 bispecific antibody.
 35. Themethod of claim 34, wherein the anti-CTLA-4 antibody product comprisesthe anti-CTLA-4 antibody or fragment thereof.
 36. The method of claim35, wherein the anti-CTLA-4 antibody is ipilimumab.
 37. The method ofclaim 33, wherein the cancer is an adenocarcinoma, acastration-resistant prostate cancer, a melanoma, a head-and-neckcancer, a lung cancer, a kidney cancer, a bladder cancer, a gastriccancer, a colorectal cancer, an ovarian cancer, a hepatocellular cancer,a hepatobiliary cancer, or a breast cancer.
 38. A method of determiningwhether cancer is amendable to treatment with an anti-CTLA-4 antibodyproduct, the method comprising: (a) isolating a population of PBMCs froma subject having cancer and a cancer-free subject; (b) quantifying thepercentages of CD4+ T_(eff) cells expressing PD-1, PD-L1, and/or CTLA-4in each of the populations; (c) comparing the percentages of CD4+T_(eff) cells expressing PD-1, PD-L1, and/or CTLA-4 in the population ofPBMCs from the subject having cancer and the percentages of CD4+ T_(eff)cells expressing PD-1, PD-L1, and/or CTLA-4 in the population of PBMCsfrom the cancer-free subject; and (d) diagnosing the subject havingcancer is amenable to treatment with an anti-CTLA-4 antibody product if:(1) the percentage of CD4+ T_(eff) cells expressing PD-1 in thepopulation of PBMCs from the subject having cancer is lower than thepercentage of CD4+ T_(eff) cells expressing PD-1 in the population ofPBMCs from the cancer-free subject; or (2) the percentage of CD4+T_(eff) cells expressing PD-L1 in the population of PBMCs from thesubject having cancer is lower than the percentage of CD4+ T_(eff) cellsexpressing PD-L1 in the population of PBMCs from the cancer-freesubject; or (3) the percentage of CD4+ T_(eff) cells expressing CTLA-4in the population of PBMCs from the subject having cancer is higher thanthe percentage of CD4+ T_(eff) cells expressing CTLA-4 in the populationof PBMCs from the cancer-free subject.
 39. The method of claim 38,wherein (1) the percentage of CD4+ T_(eff) cells expressing PD-1 in thepopulation of PBMCs from the subject having cancer is no greater thanabout 21%; or (2) the percentage of CD4+ T_(eff) cells expressing PD-L1in the population of PBMCs from the subject having cancer is no greaterthan about 23.5%; or (3) the percentage of CD4+ T_(eff) cells expressingCTLA-4 in the population of PBMCs from the subject having cancer is atleast about 15.6%.
 40. The method of claim 38, wherein the anti-CTLA-4antibody product is selected from the group consisting of an anti-CTLA-4antibody or fragment thereof, an anti-CTLA-4 chimeric antibody, ananti-CTLA-4 CDR-grafted antibody, an anti-CTLA-4 single-chain antibody,an anti-CTLA-4 single-chain variable fragment, an anti-CTLA-4 Fabantibody fragment, an anti-CTLA-4 Fab′ antibody fragment, an anti-CTLA-4F(ab′)2 antibody fragment, an anti-CTLA-4 bi-body, an anti-CTLA-4tri-body, an anti-CTLA-4 diabody, and an anti-CTLA-4 bispecificantibody.
 41. The method of claim 39, wherein the anti-CTLA-4 antibodyproduct comprises the anti-CTLA-4 antibody or fragment thereof.
 42. Themethod of claim 41, wherein the anti-CTLA-4 antibody is ipilimumab. 43.The method of claim 38, wherein the cancer is an adenocarcinoma, acastration-resistant prostate cancer, a melanoma, a head-and-neckcancer, a lung cancer, a kidney cancer, a bladder cancer, a gastriccancer, a colorectal cancer, an ovarian cancer, a hepatocellular cancer,a hepatobiliary cancer, or a breast cancer.
 44. A method of determiningwhether cancer is amendable to treatment with an anti-CTLA-4 antibodyproduct, the method comprising: (a) isolating a population of PBMCs froma subject suspected of having cancer; (b) determining the percentages ofCD4+ T_(eff) cells expressing PD-1, PD-L1, and/or CTLA-4 the populationof PBMCs; and (c) determining the subject's cancer is amendable totreatment with the anti-CTLA-4 antibody product if: the percentage ofCD4+ T_(eff) cells expressing PD-1 in the population of PBMCs from thesubject having cancer is no greater than about 21%, or the percentage ofCD4+ T_(eff) cells expressing PD-L1 in the population of PBMCs from thesubject having cancer is no greater than about 23.5%, or the percentageof CD4+ T_(eff) cells expressing CTLA-4 in the population of PBMCs fromthe subject having cancer is at least about 15.6%; or determining thesubject's cancer is not amenable to treatment with the anti-CTLA-4antibody product if: the percentage of CD4+ T_(eff) cells expressingPD-1 in the population of PBMCs from the subject having cancer isgreater than about 21%, or the percentage of CD4+ T_(eff) cellsexpressing PD-L1 in the population of PBMCs from the subject havingcancer is greater than about 23.5%, or the percentage of CD4+ T_(eff)cells expressing CTLA-4 in the population of PBMCs from the subjecthaving cancer is less than about 15.6%.
 45. The method of claim 44,wherein the anti-CTLA-4 antibody product is selected from the groupconsisting of an anti-CTLA-4 antibody or fragment thereof, ananti-CTLA-4 chimeric antibody, an anti-CTLA-4 CDR-grafted antibody, ananti-CTLA-4 single-chain antibody, an anti-CTLA-4 single-chain variablefragment, an anti-CTLA-4 Fab antibody fragment, an anti-CTLA-4 Fab′antibody fragment, an anti-CTLA-4 F(ab′)2 antibody fragment, ananti-CTLA-4 bi-body, an anti-CTLA-4 tri-body, an anti-CTLA-4 diabody,and an anti-CTLA-4 bispecific antibody.
 46. The method of claim 45,wherein the anti-CTLA-4 antibody product comprises the anti-CTLA-4antibody or fragment thereof.
 47. The method of claim 46, wherein theanti-CTLA-4 antibody is ipilimumab.
 48. The method of claim 44, whereinthe cancer is an adenocarcinoma, a castration-resistant prostate cancer,a melanoma, a head-and-neck cancer, a lung cancer, a kidney cancer, abladder cancer, a gastric cancer, a colorectal cancer, an ovariancancer, a hepatocellular cancer, a hepatobiliary cancer, or a breastcancer.